Our long-term goal is to build an HIV-resistant immune system that controls HIV-1 replication in the absence of HAART. Based on prior studies in the field, it is likely that both enhanced HIV-specific immunity, as well as a population of CD4 T cells resistant to HIV infection will be required to achieve this goal. In collaboration with Sangamo Biosciences, we recently performed a clinical trial that infused T cells rendered HIV resistant by zinc finger nuclease disruption of the CCR5 coreceptor. The viral load of one of the individuals in this study dropped below the limit of detection in the absence of HAART, suggesting that infused HIV-1 resistant CD4 T cells are capable of controlling HIV-1 replication. Another benefit of our last IPCP was the co-development (Penn and Sangamo) of a potent antiviral construct called C34-CXCR4. In this application, we will determine the clinical utility o this construct, and by comparing our clinical data from the CCR5 ZFN study, we hope to gain important insight into the key factors required to protect CD4 T cells in vivo and to better study how partial restoration of the CD4 T cell response enables control of HIV-1 replication. Traditionally, throughout our academic/industry program project grants, a consistent theme has been to concurrently test one concept in the clinic while performing research that will serve as the basis and rationale for the next clinical trial. A key component of this application is the continuation of that tradition. The elements of our proposal are: 1) A Phase I Study of C34-CXCR4 Peptide-Modified CD4 T Cells in HIV-1 (Project 1, Tebas [Penn]): This project will test the safety and feasibility of infusing autologous T cells expressing a C34-CXCR4 fusion construct in HIV-1 infected individuals. 2) C34-modified Coreceptors as Potent Trans-dominant Inhibitors of HIV-1 Entry (Project 2, Holmes [Sangamo]): This project will utilize Sangamo's zinc finger nuclease technology to target C34-CXCR4 into the CXCR4 locus in primary human T cells, limiting the genotoxicity and the possibility of vector silencing. Efforts wll also be made to understand how this construct is so effective against all strains of HIV-1 and to improve its activity if possible. 3) Designing T cells to Functionally Cure HIV-1 infection (Project 3: Riley [Penn]). This project seeks to engineer HIV-1 resistant, HIV-1 specific CD4 T cells that can control HIV-1 replication long-term in the absence of HAART in a humanized mouse model of HIV-1 infection. 4) Programming Long-Term Durable HIV-1 Specific T cell Responses (Project 4: Wherry [Penn]). This project aims to molecularly define the pathways that promote durability in human CD4 T cells using state of the art profiling technology and an array of relevant models to probe factors that enable CD4 T cells to maintain high levels of activity for prolonged periods of time. The Program is supported by 2 Cores: Core A is the administrative Core (PI, Riley); Core B is the Sequencing and Viral Evolution Core (PI, Bushman). In addition, our Program takes advantage of existing School of Medicine, ITMAT, and CFAR Cores to promote cost sharing and avoid duplication of resources.